Abstract Adults in the US are now 28 times more likely to develop melanoma than 75 years ago. Among other genetic abnormalities, mutations in the INK4a locus on chromosome 9p21 are prominent in melanoma and can be found in nearly 50% of familial cases. Two independent tumor suppressors which are transcribed from the INK4a locus, p16 (CDKN2A) and ARF (p14ARF), share common exons 2 and 3 but are translated in unique reading frames. p16 causes cell cycle and growth arrest in the G1 phase by binding cyclin-dependent kinases 4 and 6 (CDK4/6), thereby displacing Cyclin D1 and inhibiting the enzymes. ARF, meanwhile, causes cell cycle and growth arrest in the G1 and G2/M phases by binding MDM2, in turn stabilizing p53. Mutations in melanoma often affect one or both genes, suggesting p16 and ARF may play equally important roles in tumor suppression. We report a 2 bp deletion, 1 bp insertion (CC to T) in exon 2 of p16/ARF found in a melanoma cell line that alters the reading frames of both genes and creates unique proteins. One transcript initiates from the ARF promoter and codes for the first 94 amino acids of ARF before switching to the reading frame of p16 to code for another 75 residues. We named the resulting protein chimera ARF (chARF). The other transcript initiates from the p16 promoter, codes for the first 80 amino acids of p16, then switches to a non-p16/non-ARF reading frame to code for an additional 63 residues. We called this protein p16-Alternate Carboxyl Terminus (p16-ACT). Based on sequence, we hypothesized that chARF could function both as ARF and p16 since it contains the MDM2 binding domain of ARF as well as most of the CDK4/6 binding domain of p16. Meanwhile, we hypothesized that p16-ACT would have no function since it is missing most of the CDK4/6 binding region of p16. To assess the function of these proteins, we overexpressed both mutant and wild-type proteins in the p16/ARF-inactivated tumor cell line U2OS. In CDK4 immunoprecipitation assays, chARF bound CDK4, but unlike wild-type p16, did not displace Cyclin D1. p16-ACT neither bound CDK4 nor displaced Cyclin D1. chARF expression strongly activated the p53/p21 pathway and arrested U2OS cells in the G1 and G2/M phases of the cell cycle in a manner identical to wild-type ARF. p16-ACT, however, had no noticeable effect on cell cycle distribution. Finally, we looked at cell proliferation and found that chARF expression suppressed monolayer cell growth and colony formation in U2OS cells by 75-85%, similar to wild-type ARF. p16-ACT expression had a small suppressive effect on colony formation, but no effect on monolayer growth. We conclude that chARF functions as wild-type ARF but neither chARF nor p16-ACT function as wild-type p16. Citation Format: Richard T. Williams, Lisa M. Barnhill, Huan Hsien Kuo, Wen Der Lin, Alice LS Yu, Mitchell B. Diccianni. Functional analysis of a chimera of p16 and ARF: All is not lost. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5202. doi:10.1158/1538-7445.AM2013-5202